PROJECT SUMMARY/ABSTRACT Macrophages constitute a major subset of cells in the cancer tumor microenvironment (TME), but despite decades of research in murine models and in vitro studies on human blood monocyte-derived macrophages there are no reliable markers to detect the various macrophage functions in the human TME and no systematic study on human macrophage in vivo functional diversity has been performed. Current understanding of macrophage biology is based on findings from murine studies and ex vivo experiments on human blood monocyte-derived macrophages but these have not translated well into human tumor physiology. A convergence of three novel technologies (Smart-3SEQ, CIBERSORTx and MIBI) now offers an opportunity to discover novel markers by studying macrophages in vivo at the microscopic level in the actual TME rather than in a model thereof. We hypothesize that those studies do not represent the true physiological state in vivo because they lack the complex context of the human TME. New technologies will enable us to discover subtypes/phenotypes associated with different functions by directly studying human tumor samples where macrophages are known to have different activities. Our proposal represents an entirely novel approach to study TAM by comparing them in the setting of two distinct carcinomas, breast cancer and colon cancer; two tumor types in which TAM fulfill opposing functions. We will also analyze TAM at the microscopic level within distinct regions of the TME, thus identifying genes that distinguish distinct macrophage subsets based on their location within tissue. Finally, we will study changes in their expression profiles during different stages of tumor progression. This project will provide fundamental new biological insights into the diversity of macrophages in the context of human cancer. It will provide new biomarkers that can be used to provide a rational decision for the choice of novel macrophage- targeting therapy.